Clinical significance of cefazolin inoculum effect in serious MSSA infections: a systematic review

Abstract Background The cefazolin inoculum effect (CzIE) is a phenomenon whereby some MSSA isolates demonstrate resistance to cefazolin when a high bacterial inoculum is used for susceptibility testing. The clinical significance of this phenotypic phenomenon remains unclear. We conducted a systematic review to answer the following question: In patients with serious MSSA infection treated with cefazolin, does infection due to CzIE-positive MSSA isolates result in worse clinical outcomes than infection due to CzIE-negative MSSA isolates? Methods Ovid MEDLINE, Embase, Cochrane CENTRAL, medRxiv and bioRxiv were searched from inception until 12 April 2023. Studies were included if they tested for CzIE in clinical isolates from MSSA infections in humans. Two independent reviewers extracted data and conducted risk-of-bias assessment. Main outcomes were treatment failure and mortality. Pooling of study estimates was not performed given the heterogeneity of patient populations and outcome definitions. Results Twenty-three observational studies were included. CzIE presence amidst MSSA isolates ranged from 0% to 55%. There was no statistically significant mortality difference in two studies that compared MSSA infections with and without CzIE, with ORs ranging from 0.72 to 19.78. Of four studies comparing treatment failure, ORs ranged from 0.26 to 13.00. One study showed a significantly higher treatment failure for the CzIE group, but it did not adjust for potential confounders. Conclusions The evidence on CzIE is limited by small observational studies. In these studies, CzIE did not predict higher mortality in MSSA infections treated with cefazolin. Our findings do not support CzIE testing in clinical practice currently.


Background
][3] Treatment of MSSA infections entails anti-staphylococcal penicillins (e.g.cloxacillin, nafcillin) or a first-generation cephalosporin such as cefazolin. 4Although advantages of cefazolin use include more convenient dosing frequency and a reduced risk of nephrotoxicity compared with anti-staphylococcal penicillins such as cloxacillin, concerns exist regarding the presence of a cefazolin inoculum effect (CzIE). 5 When CzIE is present, it suggests the possibility of increased resistance against cefazolin when there is presence of higher bacterial burden.CzIE is an in vitro phenomenon whereby an MSSA isolate is identified as being susceptible to cefazolin when a standard bacterial concentration is tested, but resistant to cefazolin (i.e.elevation in MIC against cefazolin) when a higher bacterial concentration is used for antibiotic susceptibility testing.More specifically, CzIE is defined as a MIC ≤8 mg/L at standard bacterial inoculum of 5 × 10 5 cfu/mL, and ≥16 mg/L at a higher bacterial inoculum of 5 × 10 7 cfu/mL (i.e. the effect is proportionate to the bacterial inoculum present at site of infection). 6The CzIE phenomenon is potentially mediated by the β-lactamase enzyme (blaZ) gene encoding type A and C β-lactamases in MSSA isolates. 7Reportedly, up to 25% of MSSA isolates exhibit CzIE, based on a North American study of 305 blood culture isolates. 6he reference standard for detecting CzIE is broth microdilution, which requires technical expertise beyond that available in routine microbiology laboratories. 8In routine practice, cefazolin susceptibility is inferred from oxacillin or cefoxitin susceptibility testing, molecular detection of mecA or mecC, or PBP2a assay as per CLSI guidelines. 8Thus, routine cefazolin susceptibility testing would not detect CzIE. 8or CzIE-positive deep-seated MSSA infections (endocarditis, bone and joint infection, deep-seated abscesses, osteomyelitis or pneumonia) 9 with a high bacterial burden being treated with cefazolin, there is a theoretical concern that cefazolin will be hydrolysed due to increased production of blaZ β-lactamases leading to treatment failure. 10However, the exact mechanism of CzIE and connection with clinical outcomes when detected remains unclear at present.
To our knowledge, no systematic review has evaluated whether the CzIE is a risk factor for poor patient outcomes with cefazolin treatment.We conducted a systematic review to answer the following question: In patients with serious MSSA infections (bacteraemia, pneumonia, pleural space infection, CNS infection, endocarditis, native bone or joint infection, prosthetic joint infection or deep abscesses) who were treated with cefazolin, does infection due to MSSA isolates that show CzIE result in worse clinical outcomes than infection due to MSSA isolates without CzIE?
Secondary objectives were to describe: (i) the proportion of MSSA isolates that displayed CzIE across studies; (ii) the diagnostic testing properties of predictors for CzIE; and (iii) the comparison of outcomes in MSSA serious infections with CzIE treated with cefazolin versus an anti-staphylococcal penicillin.

Protocol and registration
This systematic review was prospectively registered on the International Prospective Register of Systematic Reviews (PROSPERO) database (CRD42023420251).

Search strategy
Studies were identified by searches across five databases: Ovid MEDLINE, Embase, Cochrane CENTRAL, medRxiv and bioRxiv.The search date range was from inception until 12 April 2023.In collaboration with a research librarian at the University of British Columbia, we developed a search strategy using relevant MeSH search terms to optimize search results (Figure S1, available as Supplementary data at JAC-AMR Online).

Eligibility criteria and exclusion criteria
We included all studies published in any language including observational studies (cohort studies, case-control studies, cross-sectional studies) and randomized controlled trials (RCTs) conducted on humans with MSSA infections or studies done on MSSA clinical isolates for which CzIE testing was done.Although this may capture non-serious MSSA infections, this strategy ensured we did not miss studies that cover our secondary objectives.
Systematic reviews, meta-analyses, case reports, commentaries, letters, abstracts, conference reports or reports of only study design were excluded.Studies that focused strictly on animal models were excluded.Studies primarily focusing on MRSA were excluded as well.If multiple published studies were based on the same patient group and reported the same mortality or treatment failure outcome, only the study describing the largest patient group was selected so that the same patient would not be double counted in the systematic review.

Data extraction
Abstracts were screened by two blinded independent authors to identify potentially relevant studies for full-text screening and review.Subsequently, two blinded independent authors read and reviewed the full text for data extraction.Disagreements during study selection and data extraction process were resolved by a third reviewer.
Data were collected on the following variables: • Journal article information: author, publication year

Outcomes
The co-primary outcomes were mortality and treatment failure, however defined by the study.Mortality could be all-cause or attributable within the time frame as reported in the study.Treatment failure could include death, complication related to infection, discontinuation of antibiotic treatment due to adverse effects, switch to another antibiotic due to lack of clinical response, and/or recurrence of infection.

Risk-of-bias assessment
Two reviewers independently assessed the risk of bias with the plan of using the tool that would be most appropriate for the study design and research question: Newcastle-Ottawa Scale (NOS) 11 for observational studies, Cochrane Risk-of-Bias (RoB) Version 2 12 for RCTs, Joanna Briggs Institute (JBI) prevalence studies checklist 13 for lab-testing studies, and QUADAS-2 tool 14 for diagnostic accuracy studies.

Statistical analysis
The proportion of CzIE in samples was calculated for each study.In a post hoc analysis, we described the prevalence of CzIE in studies categorized by country and continent.
For diagnostic parameters of CzIE predictors, we calculated the sensitivity, specificity and likelihood ratios with corresponding 95% CIs for each study in comparison with the listed reference standard (i.e.broth micro or macro-dilution).In 2 × 2 tables with a zero in one cell, 0.5 was added to all cells before doing the calculations. 15here was significant clinical heterogeneity in terms of how the patient population and outcomes were defined across studies.Therefore, only a descriptive analysis of individual studies was done.For each study, we compared the mortality and treatment failure as defined by the study between MSSA isolates with CzIE and MSSA isolates without CzIE in patients with MSSA infections who were treated with cefazolin.We calculated the unadjusted OR with MSSA isolates without CzIE as the reference and 95% CI for mortality and treatment failure.We also reported the adjusted ORs if provided by the study.
Data analysis was performed using the statistical software SPSS ver27 2020 and R.

Risk-of-bias assessment
Risk-of-bias assessment is described in Table S2 for 10 cohort studies, Table S3 for 12 lab-testing studies and Table S4 for one diagnostic accuracy study.For cohort studies, each study was rated on an overall score from 1 to 9, with 6-9 being high quality, 3-5 being fair and 0-2 being poor quality. 11Nine studies scored at least 6 points.The remaining study had a score of 4. Of note, there were no eligible RCTs and hence the RoB Version 2 tool was not applicable in our assessment.

Proportion of MSSA isolates that displayed CzIE
There were 19 studies that reported the proportion of MSSA isolates displaying CzIE as defined by MIC ≤8 mg/L at standard inoculum and MIC ≥16 mg/L at high inoculum (Table 2).The median number of isolates per study was 185 (range 51 to 690), with a median of 14.4% of isolates being positive for CzIE (range 0% to 54.5%).The proportion of CzIE in MSSA ranged from 0% to 18.7% in North American countries, 36.0% to 54.5% in South American countries, 2.5% to 11.0% in European countries, and 5.8% to 21.8% in Asian countries.

Mortality and treatment failure
Only two studies reported mortality outcomes (Table 4).The mortality definition varied in terms of timepoint, which ranged from 1 month to 3 months.The wide 95% CIs reflect small number sizes and inconclusive results.No study showed a significant difference in mortality between CzIE-positive and -negative MSSA infections.
Four studies reported treatment failure outcomes (Table 4).The definition for treatment failure varied across studies.A single study found a statistically significant increase in treatment failure for CzIE-positive isolates with an OR of 3.93 (95% CI: 1.08 to 14.31) (Table 4). 22In this study, there was no adjustment for potential confounders in the subgroup analysis when the CzIE-positive group was compared with the CzIE-negative group. 22

Cefazolin versus anti-staphylococcal penicillin for CzIE-positive MSSA infections
One study compared the mortality and treatment failure between an anti-staphylococcal penicillin and cefazolin for CzIE-positive MSSA infections. 22At the end of 1 month and 3 months, 2/13 (15.4%) patients in the cefazolin group and 1/11 (9.1%) patients in the nafcillin group died (OR 1.82; 95% CI: 0.14 to 23.26). 22Treatment failure, defined as discontinuation of antibiotics due to adverse effects, antibiotic change due to clinical failure, death within 1 month and recurrence, occurred in 8/13 (61.5%) for the cefazolin group and 4/11 (36.4%) patients for the nafcillin group (OR 2.80; 95% CI: 0.53 to 14.74).

Discussion
Our results show that the clinical impact of CzIE is based on small observational studies that provide poor quality evidence.Overall, the proportion of MSSA infections displaying CzIE ranged from 0% to 55% across studies.No study found a significant difference in mortality between CzIE-positive and -negative MSSA infections treated with cefazolin.All but one study found no significant difference in treatment failure between CzIE-positive and -negative MSSA infections treated with cefazolin.Therefore, there is a lack of evidence to support that CzIE is clinically important currently.
Strengths of our review included a comprehensive search of multiple databases that included preprints and had no language restrictions.There was also rigorous screening and data collection by two independent reviewers for each study.
Limitations included the very limited sample size pool of four studies that reported mortality and treatment outcomes. 7,10,19,22ost studies did not adjust for potential confounders.There was significant heterogeneity across studies for types of infections and definition of outcomes.Only one study reported clinical outcomes for anti-staphylococcal penicillins. 22It is important to compare the effectiveness of cefazolin versus anti-staphylococcal penicillin in the treatment of MSSA infection  MSSA isolates that display CzIE may also have other intrinsic bacterial characteristics that make the isolates more virulent and the infection more deadly regardless of antibiotic treatment choice.
If that is the case, then MSSA with CzIE treated with cefazolin versus an anti-staphylococcal penicillin would have similar outcomes.
Large studies are needed to provide higher quality evidence on whether CzIE is clinically important.The ideal study should include serious and deep-seated infections in which CzIE may be clinically relevant.MSSA infections with CzIE treated with cefazolin should be compared with MSSA infections without CzIE treated with cefazolin as well as MSSA infections with CzIE treated with an anti-staphylococcal penicillin.Lastly, adjustment should be made for potential confounders.

Conclusions
In conclusion, there is very low quality of evidence at present that does not support the theory that CzIE translates to worse outcomes in terms of mortality or treatment failure for serious MSSA infections being treated with cefazolin.Thus, our study supports the CLSI recommendation that CzIE should not be tested in clinical settings outside of research until there is more evidence to suggest otherwise. 35,36Clinical microbiology laboratories should avoid routine testing for CzIE when pursuing microbiological workup of MSSA clinical isolates, because the current evidence does not support the use of CzIE results when making clinical treatment decisions for MSSA infections.Systematic review

Identification of studies via databases and registers Identification Screening Included Figure 1.
Three studies reported predictors for CzIE that allowed calculation of diagnostic properties.Surrogate predictors included Flow diagram for inclusion of studies.

Table 3 .
The rapid colorimetric test specifically designed for detection of CzIE 28 had the best combination of sensitivity and specificity based on the point estimate and CI.

Table 2 .
Proportion of MSSA isolates that tested positive for cefazolin inoculum effect (CzIE) in studies categorized by country and continent a a Study reference 10 was not included because the country of origin for the MSSA isolates was unclear.In this study, 19/98 (19.2%) displayed CzIE.Study reference 20 was not included because the testing method and definition for CzIE are different from our definition.Study reference 21 was not included because it tested only MSSA isolates that tested positive for type A blaZ gene.

Table 3 .
Diagnostic properties of surrogate predictors for cefazolin inoculum effect (CzIE) NLR, negative likelihood ratio; PLR, positive likelihood ratio; Sn, sensitivity; Sp, specificity.Systematic review with CzIE.The rationale is that if MSSA infections with CzIE treated with cefazolin had worse outcomes than MSSA infections without CzIE treated with cefazolin, there may be a reason other than cefazolin treatment and CzIE for the difference in outcome.

Table 4 .
Clinical outcomes comparing CzIE-positive versus CzIE-negative isolates for MSSA infections treated with cefazolin odds ratio, otherwise OR refers to unadjusted odds ratios; CzIE, cefazolin inoculum effect.